This proposal concerns molecular mechanisms leading to the development of neural crest cells. The neural crest is a population of stem-cell-like cells, which form in vertebrate embryos at the neural plate border and migrate to diverse locations in the body to give rise to diverse cell types, including face cartilage, melanocytes and the peripheral nervous system. Neural crest forms as a result of inductive interactions of neuroectoderm, epidermal ectoderm and the underlying mesoderm. One embryonic signaling pathway that plays an essential role in neural crest induction is the Wnt pathway, known to control cell proliferation, fates, cell polarity and motility in many cell types. Whereas the role for the Wnt/2-catenin pathway in neural crest regulation has been established, the functions of 2-catenin-independent Wnt signals in this process remain largely unknown. Our preliminary studies reveal a function for 2-catenin-independent Wnt proteins in neural crest specification. We have also discovered that PAR proteins are involved in the activation of neural crest-specific genes. To connect cell polarization to neural crest specification, we plan to investigate the mechanism, by which PAR proteins influence neural crest fates. The proposed studies will evaluate at which developmental step PAR function is required, whether the response to known neural crest regulators is altered and whether the proper subcellular localization of PAR proteins is critical for this process. Using an unbiased protein interaction screen and the candidate protein approach, we will identify downstream targets of PAR proteins, which contribute to neural crest development. Other studies will determine how upstream signaling pathways regulate PAR localization, levels and activity to specify neural crest fates. These experiments will be carried out in Xenopus embryos, which represent a rapid in vivo system for gene function and allow classical cell biological and biochemical experiments. By connecting PAR and Wnt signaling during the initial stages of craniofacial development, the proposed experiments will contribute to the understanding of both the basic developmental mechanisms and will be relevant to the disease. A large number of human diseases, such as craniosynostosis, Waardenburg and Hirschsprung's syndromes, and cancer, have been associated with neural crest abnormalities. Studying the mechanisms underlying the development of the neural crest cells should provide insights into human diseases associated with stem cell disorders and cancer. PUBLIC HEALTH RELEVANCE: This application concerns molecular mechanisms leading to the development of neural crest cells. Since neural crest abnormalities are known to lead to craniosynostosis, Waardenburg and Hirschsprung's syndromes, and cancer, the proposed studies are highly relevant to human diseases associated with stem cell disorders and tumorigenesis.